wi-gatk

The new GATK-based pipeline for wild isolate Caenorhabditis strains

Pipeline Overview

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To run the pipeline:

nextflow main.nf --help
nextflow main.nf --debug
nextflow main.nf --sample_sheet=/path/sample_sheet.txt --species c_elegans --bam_location=/path/to/bams --gvcf_location=/path/to/gvcfs

    parameters                 description                           Set/Default
    ==========                 ===========                           ========================
    --debug                    Use --debug to indicate debug mode    false
    --species                  Species to call variants from         null
    --sample_sheet             Sample sheet                          null
    --bam_location             Directory of BAM files                {dataDir}/{species}/WI/alignments
    --gvcf_location            Directory of gVCF files               {dataDir}/{species}/WI/gVCFs
    --mito_name                Contig not to polarize hetero sites   MtDNA
    --partition                Partition size in bp for subsetting   1000000
    --gvcf_only                Create sample gVCFs and stop          false
    --split_samples            Create individual sample vcfs         false
    --username                                                       {user}

    Reference Genome
    --------------- 
    --reference                The fa.gz reference file to use       {dataDir}/{species}/genomes/{project}/{ws_build}/{species}.{project}.{ws_build}.genome.fa.gz

    Variant Filters         
    ---------------           
    --min_depth                Minimum variant depth                 3
    --qual                     Variant QUAL score                    30.0
    --strand_odds_ratio        SOR_strand_odds_ratio                 5.0
    --quality_by_depth         QD_quality_by_depth                   20.0
    --fisherstrand             FS_fisher_strand                      100.0
    --high_missing             Max % missing genotypes               0.95
    --high_heterozygosity      Max % max heterozygosity              0.10

Software Requirements

• The latest update requires Nextflow version 24.10.0+. On Rockfish, you can access this version by loading the nf24_env conda environment prior to running the pipeline command:

ml anaconda
conda activate /data/eande106/software/conda_envs/nf24_env

Typical use for debugging:

nextflow run -latest andersenlab/wi-gatk --debug

Typical use for new bam files:

nextflow run -latest andersenlab/wi-gatk --sample_sheet=/path/sample_sheet_GATK.txt --species c_elegans --bam_location=/path/to/bams --gvcf_location=/path/to/gvcfs

Parameters

-profile ( optional )

default = rockfish

There are three configuration profiles for this pipeline.

• rockfish - Used for running on Rockfish
• quest - Used for running on Quest
• local - Used for local development

--sample_sheet

The sample sheet is a list of strains to jointly call variants from, one strain per line

AB1
AB4
BRC20067

--bam_location (optional if model worm species specified)

Path to directory holding all the alignment files for strains in the analysis. Defaults to /vast/eande106/data/{species}/WI/alignments/

--gvcf_location (optional if model worm species specified)

Path to directory holding all the gVCF files for strains. If one or more strain doesn't have a gVCF file, for example a new strain, one is created. Defaults to /vast/eande106/data/{species}/WI/alignments/

--species

default = null

Options: c_elegans, c_briggsae, or c_tropicalis (for default reference, bam, and gvcf paths) or any other with reference, bam directory, and gvcf directory specified

--project (optional, only used with model worm species)

default = PRJNA13758

WormBase project ID for selected species. Choose from some examples here

--ws_build (optional, only used with model worm species)

default = WS283

WormBase version to use for reference genome.

--reference (optional if model worm species specified)

A fasta reference indexed with BWA. On Rockfish, the reference is available here:

/vast/eande106/data/c_elegans/genomes/PRJNA13758/WS283/c_elegans.PRJNA13758.WS283.genome.fa.gz

Note

If running on Rockfish, instead of changing the reference parameter, opt to change the species, project, and ws_build for other reference build (and then the reference will change automatically)

--mito_name (optional)

default = MtDNA

Name of contig to skip het polarization. Might need to change for other species besides c_elegans if the mitochondria contig is named differently

--partitions (optional)

default = 1000000

Size in bp for partitioning the genome for subprocessing genotyping and filtering

--gvcf_only (optional)

default = false

Run only the gVCF creation steps of the workflow

--split_samples (optional)

default = false

Create individual vcf files, one per sample, from the hard-filtered vcf file

-output-dir (optional)

default = WI-{today's date} where the date is formatted as YYYYMMDD

A directory in which to output results

Note

This option is a nextflow parameter and so only uses a single dash to specify it

Output

The final output directory looks like this:

├── variation
│   ├── *.hard-filter.vcf.gz
│   ├── *.hard-filter.vcf.tbi
│   ├── *.hard-filter.stats.txt
│   ├── *.hard-filter.filter_stats.txt
│   ├── *.soft-filter.vcf.gz
│   ├── *.soft-filter.vcf.tbi
│   ├── *.soft-filter.stats.txt
│   ├── *.soft-filter.filter_stats.txt
│   └── strain_vcf
│       ├── *.vcf.gz
│       └── *.vcf.gz.tbi
└── report
    ├── multiqc.html
	└── multiqc_data
        └── multiqc_*.json
   

Relevant Docker Images

• quay.io-biocontainers-samtools-1.21--h50ea8bc_0 (link): Docker image maintained by biocontainers for samtools
• quay.io-biocontainers-bcftools-1.16--hfe4b78e_1 (link): Docker image maintained by biocontainers for bcftools
• quay.io-biocontainers-gatk4-4.6.1.0--py310hdfd78af_0 (link): Docker image maintained by biocontainers for gatk
• quay.io-biocontainers-multiqc-1.8--py_1 (link): Docker image maintained by biocontainers for multiqc
• andersenlab-hetpolarization-1.10 (link): Docker image is created within this pipeline using GitHub actions. Whenever a change is made to env/hetpolarization.Dockerfile or .github/workflows/build_docker.yml GitHub actions will create a new docker image and push if successful
• andersenlab-r_packages-v0.7 (link): Docker image is created manually, code can be found in the dockerfile repo.

Make sure that you have followed the Nextflow configuration described in the dry-guide prior to running the workflow.